PROJECT SUMMARY Drug-resistant tuberculosis (TB) is a major global epidemic and poses a particular threat to HIV-infected individuals. With few effective drugs available for treatment, multidrug- and extensively drug-resistant (M/XDR) TB carry a high mortality rate and threaten global TB and HIV control efforts. M/XDR TB treatment is long (18- 24 months) and associated with serious side effects. As a result, 10-25% of patients prematurely interrupt therapy, placing them at increased risk for treatment failure, acquisition of additional resistance, and death. New and repurposed medications have recently been found to improve survival and cure rates. Bedaquiline, the first new TB drug in 40 years, and clofazimine?a drug primarily used for leprosy?have been at the center of this treatment revolution. In 2018, WHO recommended that all MDR and XDR TB treatment regimens include bedaquiline and clofazimine. Bedaquiline, however, has an extremely long half-life (5.5 months). Patients who interrupt therapy are likely exposed to bedaquiline monotherapy as companion drugs are more rapidly eliminated?placing them at high risk of developing bedaquiline resistance. Clofazimine, which has a half-life of 70 days, may protect against resistance as a companion drug, but cross-resistance between bedaquiline and clofazimine may develop. Given the high frequency of treatment interruption, there is danger that as bedaquiline and clofazimine are scaled up worldwide, resistance may become widespread. In the proposed study, we seek to understand the complex interplay of bedaquiline and clofazimine?s pharmacokinetics (PK) on the risk of resistance during treatment interruption. We will enroll MDR and XDR TB patients who have returned to care after interrupting a bedaquiline- and clofazimine-containing regimen. In Aim 1, we will characterize the presence of phenotypic and genotypic drug resistance to bedaquiline and clofazimine to examine how the duration of interruption affects the risk of resistance. In Aim 2, we will measure plasma concentrations of bedaquiline and clofazimine and intracellular bedaquiline concentrations after treatment interruption. We will use population PK modeling to understand the pharmacokinetics of these drugs during interruption and determine the concentrations associated with phenotypic or genotypic resistance. In Aim 3, we will use a novel, deep sequencing assay to identify minority resistant subpopulations (i.e., microheteroresistance) which are not detectable by phenotypic testing or whole genome sequencing. We will follow subjects for 6 months to determine if heteroresistance predicts treatment failure or acquired resistance. South Africa has among the highest global burden of drug-resistant TB and HIV and has initiated >21,000 patients on bedaquiline to date. The aims of this study will answer fundamental questions about bedaquiline and clofazimine pharmacology and resistance that will directly inform their use in South Africa and worldwide. Our study will draw from the largest bedaquiline treatment program in the world and addresses research priorities outlined by the NIH and the US Federal TB Task Force.